A basic element of the practice of therapeutics is the administration of active agents to subjects of treatment. In many situations the problem of administration is one of delivering enough of an active agent in an efficient manner to the sight of action. Liposome encapsulation has, in some instances, offered effective delivery of active agents. However particular active agents have not been efficiently delivered or not delivered in high enough concentrations or in concentrations relative to accompanying lipid that entailed excessive lipid administration. Thus a method of forming a high ratio active agent:lipid complex is of great interest. Active agents (also termed bioactive agents) are for example, drugs hormones, proteins, dyes, vitamins, or imaging agents. As used in the present invention, the term active agent is understood to include any compound having biological activity, e.g., drugs and other therapeutic agents such as peptides, hormones, toxins, enzymes, neurotransmitters, lipoproteins, glycoproteins, immunomodulators, immunoglobulins, polysaccharides, cell receptor binding molecules, nucleic acids, polynucleotides, and the like, as well as including biological tracer substances such as dyes, radio-opaque or radio-contrast agents for X-ray imaging (collectively "contrast agents"), and fluorescent agents.
Prophylactic use of antimicrobial agents is a widely discussed course of pharmaceutical treatment, e.g., "Prophylactic Use of Antimicrobial Agents in Adult Patients", Mayo Clin Proc, 62:1137-1141 (1987). In appropriate cases, rheumatic fever, bubonic plague, malaria and other diseases are treated prophylactically. A particularly well recognized area of antimicrobial, and especially antibacterial, prophylaxis is the preoperative use of antimicrobial agents. Generally, it has been a concomitant of surgical prophylaxis that the antimicrobial agent be given only during or at most no more than about three hours before surgery.
A central consideration in the administration of prophylactic drug therapy is the ability and ease with which a "minimum inhibitory concentration" ("MIC") of a particular antimicrobial agent may be established and the interval over which such level may be maintained in a subject animal. An MIC is defined as the lowest level of a particular antimicrobial agent (whether antibacterial, anti-infective or other) that will prevent proliferation of the pathogenic organism being treated. An MIC may be established in a physiological fluid such as cerebro-spinal fluid, serum or plasma or in tissue, and particularly tissue at the site of infection (such as the reticulo-endothelial system in disseminated infections).
Conventionally, the MIC for a particular organism is measured in an in vitro system, and is expressed in terms of the amount of drug (by weight) per amount (usually volume) of material in which the drug is dispersed. For a drug to exert a therapeutic or prophylactic effect in vivo the concentration of such drug in vivo will usually be required to be about equal to or greater than the MIC as measured in vitro. Furthermore, the greater the concentration of drug above the MIC and the greater the period of time the in vivo drug level exceeds the MIC, the greater the therapeutic response.
To express the parameter of time over which an in vivo plasma concentration of antibiotic equals or exceeds the MIC of a challenging pathogen the term T greater than MIC.sub.p will be used and written as T.sub.p.
To express the parameter of time over which an in vivo tissue concentration of antibiotic equals or exceeds the MIC of a challenging pathogen the term T greater than MIC.sub.t will be used and written as T.sub.t.
Liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous volume. Liposomes may be unilamellar vesicles (possessing a single bilayer membrane) or multilameller vesicles (onion-like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer). The bilayer is composed of two lipid monolayers having a hydrophobic "tail" region and a hydrophilic "head" region. The structure of the membrane bilayer is such that the hydrophobic (nonpolar) "tails" of the lipid monolayers orient toward the center of the bilayer while the hydrophilic "heads" orient toward the aqueous phase.
The original liposome preparation of Bangham, et al. (J. Mol. Biol., 1965, 13:238-252) involves suspending phospholipids in an organic solvent which is then evaporated to dryness leaving a phospholipid film on the reaction vessel. Next, an appropriate amount of aqueous phase is added, the mixture is allowed to "swell," and the resulting liposomes which consist of multilamellar vesicles (MLVs) are dispersed by mechanical means. This technique provides the basis for the development of the small sonicated unilamellar vesicles described by Papahadjopoulos et al. (Biochim. Biophys. Acta., 1968, 135:624-638), and large unilamellar vesicles.
Unilamellar vesicles may be produced using an extrusion apparatus by a method described in Cullis et al., PCT Application No. WO 86/00238, published Jan. 16, 1986, entitled "Extrusion Technique for Producing Unilamellar Vesicles" incorporated herein by reference. Vesicles made by this technique, called LUVETS, are extruded under pressure through a membrane filter. LUVETs, being usually of about 500 nm diameter or less, and frequently about 100 nm, are preferred liposomes of the instant invention. LUVETs will be understood to be included in the term "unilamellar vesicle".
Another class of liposomes are those characterized as having substantially equal lamellar solute distribution. This class of liposomes is denominated as stable plurilamellar vesicles (SPLV) as defined in U.S. Pat. No. 4,522,803 to Lenk, et al., monophasic vesicles as described in U.S. Pat. No. 4,588,578 to Fountain, et al. and frozen and thawed multilamellar vesicles (FATMLV) wherein the vesicles are exposed to at least one freeze and thaw cycle; this procedure is described in Bally et al., PCT Publication No. 87/00043, Jan. 15, 1987, entitled "Multilamellar Liposomes Having Improved Trapping Efficiencies". The teachings of these references as to preparation and use of liposomes are incorporated herein by reference.
Liposomes with a diameter of about 500 nm or less are termed "small liposomes". Similarly, the cephalosporin:lipid complexes of this invention will be termed "small" at a diameter of about 500 nm or less. Diameter in describing a population of liposomes or vesicles will be understood to reflect a range of diameters.
The cephalosporin:lipid complexes of this invention are in the form of liposomes at lower drug to lipid ratios. At higher drug to lipid ratios the complexes appear to retain the bilayer organization of liposomes but, in electronmicrograph, may be other than completely closed. The term "complex" is to be understood to encompass both liposomes as well as the higher ratio cephalosporin:lipid entities even if such entities differ from liposomes in having incompletely closed bilayers or other anomalies.
Iodinated contrast agents also form complexes with lipids as an aspect of this invention and exhibit high capture volumes of up to about 20 ul/umole lipid. These appear to be in the form of liposomes and are predominately unilamellar and uniform in size at about 0.5 microns. Prior to this invention, contrast agent:lipid ratios of about 2.7:1 were reported as to non-ionic iodinated contrast agents and 1:1 as to negatively charged iodinated contrast agents (e.g., diatrizoate).
Over the past ten years, there have been several groups attempting to develop particulate contrast agents which would specifically opacify organs of the RE system, Seltzer, S. E., Liposomes as Drug Carriers, Gregoriadis, G., ed., pp 509-525 (John Wiley and Sons, Ltd. New York 1988). One such application involves the detection of metastatic lesions in the liver by x-ray computed tomography (CT). Conventionally a patient is dosed with a water soluble contrast agent before CT scanning, but because the agent has essentially equal access to both normal and tumor tissue, there is minimal if any contrast between the two tissues. However, if a patient were dosed with particulate contrast agent, the Kupffer cells of the liver would phagocytose the agent causing the cells to become opaque leaving the tumor cells dark. Several particulate agents have been developed and used in humans but these agents have some associated toxicities presumably due to their extended tissue residence time. Particulate contrast agents are reported in Cohen, Z., et al J. Comput. Assist. Tomogr., 5:843-7 (1981); Miller, D. L., et al., AJR, 143:235-243 (1984); Longino, M. A., et al., J. Comput. Assist. Tomogr., 7:775-9 (1983); Mattrey, R. F., et al., Radiology, 145:755-8, (1983); Violante, M. R., et al., Invest. Radiol., 16:40-5 (1981). Another means of achieving a particulate contrast agent is entrapment of the agent in liposomes, Havron, H. A., et al., Radiology, 140:507-11 (1981). The lipid dose required by these preparations is too high for application to humans. The highest reported iodine:lipid ratio in the literature for diatrizoate, the most commonly used contrast agent, is about 1:1 (wt/wt).
In some applications admixing a lipid with cholesterol will reduce the entrapment of cephalosporin. This is potentially the result of cholesterol dispersing in the lipid bilayer and decreasing bilayer rigidity. Lipids are characterized in having a hydrophobic "tail" region and a hydrophilic "head" region. The structure of the membrane bilayer is such that the hydrophobic (nonpolar) "tails" of the lipid monolayers orient toward the center of the bilayer while the hydrophilic "heads" orient toward the aqueous phase. The tail region may be comprised of fatty acyl chains (also denoted as carbon chains), or a single such chain as in the case of alpha-tocopherol hemi-succinate. Examples of lipids are the phospholipids such as phosphatidylcholine (PG) (egg phosphatidylcholine is denoted EPG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylinositol (PI), sphingomyelin (SPM), and the like, alone or in combination and particularly in hydrogenated or saturated form of the carbon chain. The phospholipids can be synthetic or derived from natural sources such as egg or soy. Synthetic phospholipids include dymyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG). The preferred lipids of this invention are those with saturated carbon chains of at least about 16 carbon units as well as nonphospholipids such as digalactosyldiglyceride. They may also contain organic acid derivatives of sterols such as cholesterol hemisuccinate (CHS), and the like. Organic acid derivatives of tocopherols may also be used as liposome-forming ingredients, such as alpha-tocopherol hemisuccinate (THS). Both CHS- and THS-containing liposomes and their tris salt forms may generally be prepared by any method Blown in the art for preparing liposomes containing these sterols. In particular, see the procedures of Janoff et al., U.S. Pat. No. 4,721,612 issued Jan. 26, 1988, entitled "Steroidal Liposomes," and Janoff et al., PCT Publication No. WO87/02219, published Apr. 23, 1987, entitled "Alpha-Tocopherol Based Vesicles," filed Sep. 24, 1986, respectively, corresponding to U.S. Pat. No. 4,861,580, issued Aug. 29, 1989. Additional known lipids are glycolipids.